Despite costly attempts to reduce bacterial contamination of water, meat, and produce, Shiga toxin-producing E. coli (STEC) and related enteric pathogens (e.g. Salmonella, Shigella, Yersinia) are causing increasingly frequent outbreaks of food borne diarrheal disease, thus constituting enormous health burdens. Each of these pathogens uses a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While T3SS effectors clearly play essential roles in bacterial virulence, their biochemical mechanisms are incompletely characterized. We are characterizing mammalian signal transduction pathways targeted by STEC effectors. We have recently described an STEC effector protein named NleH1 that inhibits the host innate immune response to infection by targeting the NF-KB transcription factor using a unique mechanism. The NF-KB family of transcription factors regulates the activation of many crucial pro-inflammatory host defenses to microbial pathogens. The activity of NF-KB at key genes involved in the innate response to bacterial infection is regulated by ribosomal protein S3 (RPS3), which possesses an accessory nuclear function as an NF-KB subunit. We discovered that NleH1 inhibits the nuclear import of RPS3, leading to the selective loss of NF-KB activity. We found that NleH1 prevents the nuclear translocation of RPS3 by inhibiting the Ik kinase complex (IKK) from phosphorylating RPS3, which is required for its nuclear translocation. The STEC serotypes that are most commonly implicated in causing deadly outbreaks of hemorrhagic colitis in humans also encode a homologous effector named NleH2. Despite sharing 84 % identity with NleH1, NleH2 stimulates rather than inhibits RPS3/NF-KB-dependent transcription. The central hypothesis for the proposed research is that the STEC NleH1 and NleH2 effectors promote bacterial survival by subverting the regulation of inflammatory responses to infection controlled by RPS3/NF-KB. The following specific aims are designed to define the molecular mechanism of these novel proteins: Aim 1. Characterize the molecular mechanism by which NleH1 inhibits IKK phosphorylation of RPS3 to prevent its nuclear import. Aim 2. Elucidate mechanistic differences between NleH1 and NleH2 and their pathophysiological significance in regulating RPS3/ NF-KB-dependent signaling. Aim 3. Quantify the importance of NleH effectors to bacterial virulence and transmission using animal models of bacterial diarrheal disease. Successful completion of the proposed research will 1) reveal how this novel group of bacterial effectors selectively modulates innate immunity; 2) clarify how pathogens have evolved to co-opt the accessory nuclear functions of ribosomal proteins; and 3) characterize how bacteria have integrated their virulence proteins into subverting host signal transduction cascades.